Field of the Invention and Related Art Statement
The present invention relates to a method of effecting an immunoassay by using affinity chromatography.
The immunoassay using the antigen-antibody reaction may be roughly classified into a labeling immunoassay using labeling substances and a non-labeling immunoassay in which the antigen-antibody complex or conjugate is directly measured without using the labeling substances. These immunoassays may be further classified into various methods. For instance, the labeling immunoassay may be classified into radio immunoassay, enzyme immunoassay, fluorescence immunoassay and others. Further, in accordance with the manner of reaction, the immunoassay may be classified into a competitive method and a non-competitive (sandwich) method. Moreover, the immunoassay may be further classified into a heterogeneous method and a homogeneous method. In the heterogeneous method, a so-called B-F separation is required for separating an immuno complex (Bound) which is produced as the result of the antigen-antibody reaction and non-reacted antigen or antibody (Free).
Among the above mentioned various methods, the heterogeneous method has recently become predominant. As one of the heterogeneous methods, there has been developed an immunoassay using affinity chromatography in which successive samples are injected into a reusable column packed with ligands such as antibody or antigen attached to a solid support to effect the antigen-antibody reaction in the column, and after the reaction, the bound antigen or antibody is measured with the aid of labeling substances and then the bound antigen or antibody is eluted or dissociated with the aid of an eluent.
The above mentioned immunoassay using the affinity chromatography has been disclosed in greater detail in "ANALYTICAL CHEMISTRY", Vol. 57, No. 14, DECEMBER 1985, pp 2754 to 2756. In this known method, in order to measure mouse anti-bovine IgG contained in a sample, Reactigel-6X (Pierce Chemical Co., Rockford) having bovine IgG applied thereto is charged in a microreactor. Then, the sample (mouse anti-bovine IgG), a labeling antibody (anti-mouse IgG-glucose oxidase) and a substrate (glucose) are successively injected into the microreactor, and an amount of produced hydrogen peroxide is measured by means of an electric chemical measuring device such as a thin-layer amperometric detector to derive an amount of the mouse anti-bovine IgG contained in the sample. After the measurement for a sample is done, a disruption buffer of pH 2.0 is injected into the column to elute the bound complexes. Next, an assay buffer of pH 6.8 of a next sample is injected into the microreactor.
In the known immunoassay method explained above, various timings during the measurement are fixedly determined as illustrated in FIG. 4 of said article. If bound complexes or conjugates could not be sufficiently eluted, there might be produced undesired carry-over of sample, and measurement error might be introduced. Therefore, eluting and reproducing time periods have to be made long. However, if a sample has an extremely high concentration, the carry-over could not be removed sufficiently. Moreover, if a sample concentration is low, the eluting and regenerating time periods would be considered unnecessarily long, and the efficiency of the process would become low.
As explained above, in the immunoassay using affinity chromatography, undesired carry-over might be introduced among samples unless the bound antigen or antibody is sufficiently eluted. The carry-over would be remarkable after the measurement of samples having high concentrations. That is to say, after a high concentration sample is measured, when a low concentration sample is analyzed, a blank value might be erroneously judged as a measured value. The above mentioned problem could be avoided or mitigated by prolonging the eluting period sufficiently. However, an elution rate (an amount of eluted substance per unit time) becomes slow in accordance with the progress of the elution, and thus in order to effect a sufficient elution, the elution time period has to be set very long, so that the efficiency of process might be decreased.